Sodium–calcium exchanger isoform-3 targeted Withania somnifera (L.) Dunal therapeutic intervention ameliorates cognition in the 5xFAD mouse model of Alzheimer’s disease

The third isoform of the Na+–Ca2+ exchanger (NCX3) is crucial for a physiological fine-tuning of the Ca2+ fluxes in excitable tissues. In this view, the NCX3 accounts for the aberrant Ca2+ influx seen during neuronal excitotoxicity, such as in Alzheimer’s disease (AD). However, little is known about NCX3 regulation and functional properties. Withania somnifera (L.) Dunal (W. somnifera), a traditional indigenous plant widely recognized for having numerous medicinal values, was undertaken to determine its potential therapeutic benefit against aggregated Aβ1–42-induced NCX3 dysregulation and the thereof cognition impairment in 5xFAD mice. The undertaken sourced dried roots of authenticated W. somnifera physicochemical compositional tests satisfied standards of pharmacognostic quality, and further phytochemical analysis of the roots methanol extract revealed the roots constitute several antioxidants. Following an intra-gastric gavage administration of synthesized W. somnifera roots methanolic extract from postnatal day 30 (P30) to P75, in vivo cognitional studies and then neurochemical examinations of the NCX3 expression level, Aβ plaque deposition, and antioxidant activities in the AD-associated brain regions of 4-month-old 5xFAD mice suggests that the oxidative stress normalizing effects of W. somnifera constituents, operating on the NCX3, may have a therapeutic role in the improvement of cognition in AD.

www.nature.com/scientificreports/ Moreover, loss on drying, total ash, water soluble ash, acid insoluble ash, water soluble extractive value, and alcohol soluble extractive value were determined as described in the Ayurvedic Pharmacopoeia of India 34 .
Preparation of W. somnifera methanol extract. The roots powder was soaked in methanol (25 g/250 ml) for 24 h withheld in a gently shaking apparatus at room temperature for extraction. The extract was filtered with a Whatman filter paper (125 mm; 1441-125, GE Healthcare Life Sciences, UK) while excess solvent was allowed to evaporate under reduced pressure at 40 °C using a rotary vacuum evaporator. Following to determining the mean yield as a mass of the obtained extract per 100 g of W. somnifera roots, the filtrate was stored in a refrigerator at 4 °C and used throughout the study.

Phytochemical screening.
Qualitative phytochemical studies of the extract (Table 1) were performed as per standard procedures [36][37][38] to determine the presence of biochemically active constituent organic compounds.
In brief, to test for the presence of alkaloids compound, few drops of iodine solution in potassium iodide were added to 2 ml of the extract solution. Formation of reddish-brown precipitate confirms the presence of alkaloids.
To test for flavonoids, 1 ml of the extract was treated with 0.5 ml of concentrated hydrochloric acid and then warmed on a water bath for 15 min. An intense red color signifies the presence of flavonoids. To test for glycosides, 50 mg of the extract was hydrolyzed with concentrated hydrochloric acid for 2 h on a water bath, filtered, and then 2 ml of the filtered hydrolysate was thoroughly mixed with 3 ml chloroform. A pink color signifies the presence of glycosides. To test for phenols, the extract (50 mg) was dissolved in 5 ml of distilled water, and to this 3 ml of 10% lead acetate solution was added. A bulky white precipitate indicates the presence of phenols. To test for tannins, 50 mg of the extract was dissolved in 5 ml of distilled water, and to these 2 drops of neutral 5% ferric chloride solution were added. A dark green color indicates the detection of tannins. To test for triterpenoids, the extract (50 mg) was treated with 3 ml chloroform, and then filtered and added with 2 drops of concentrated sulphuric acid, gently shaken and then allowed to stand. The appearance of golden yellow color indicates the presence of triterpenoids. To test for the presence of phytosterols, 2 drops of concentrated sulphuric acid were slowly added to the extract (50 mg) dissolved in 2 ml acetic anhydride. Formation of a brown ring at the junction confirms the presence of phytosterols. For the presence of saponins, 50 mg of the extract was diluted with distilled water to make up to 20 ml in a graduated cylinder and shaken for 15 min. Formation of a 2 cm layer of foam indicates the presence of saponins. After the qualitative phytochemical analysis, the determined constituents of the extract that are defined to contribute towards the overall antioxidant activities were quantitated ( Table 2) according to standard operating methods 39 , viz. using gravimetric analysis for alkaloids, quercetin standard curve (y = 0.012x + 0.0604; R 2 = 0.9 918) for flavonoids, gallic acid curve (y = 0.0072x + 0.0472; R 2 = 0.9897) for phenols, and tannic acid curve (y = 0.0061x + 0.0434; R 2 = 0.9895) for tannins. In brief, for quantification of alkaloids, 50 mg of the dried sample was gently mixed with 200 ml of 10% acetic acid in ethanol and allowed to stand for 4 h. The mixture was then concentrated on a water bath for up to one-third of its original volume followed by the addition of ammonium hydroxide to the mixture dropwise until it formed precipitate. After 3 h of the mixture sedimentation, the precipitate was washed with ammonium hydroxide (0.1 M) and then filtered. The filtrate (alkaloids) was dried in an oven and quantitated as a percentage of the dried sample. For the total flavonoid content, 0.3 ml of the extract was mixed with 0.15 ml of sodium nitrite (0.5 M) and 0.15 ml of aluminum chloride hexahydrate (0.3 M) and allowed to stand for 1 h. Then sodium hydroxide was added to the mixture and, after 5 min of mixing, absorbance was recorded at 560 nm. Total flavonoid content was determined using quercetin standard curve as µg quercetin equivalents/mg of the extract. For total phenolic content, 1 ml of the extract was mixed with 9 ml of distilled www.nature.com/scientificreports/ water. To the mixture, 1 ml of Folin-Ciocalteu's phenol reagent followed by 10 ml of sodium carbonate solution were added and the total volume was made to 25 ml by adding distilled water and left to react for 90 min. The optical density was determined at 750 nm and the quantity of total phenolic was calculated using the gallic acid curve as µg gallic acid equivalent/mg of the extract. For quantification of tannins, 5 ml of the extract was mixed with 2 ml of ferric chloride in hydrochloric acid (0.1 N) and potassium ferricyanide (0.008 M) and left to react for 10 min. Absorbance was taken at 120 nm and the quantity of tannins was determined using the tannic acid curve as µg tannic equivalent/mg of the extract.
Animals. This study used a cohort of 1-4-month-old, 48 5xFAD 32 male mice purchased from the Experi- Animals grouping and treatment. Male 5xFAD mice (1-month-old, weighing 16.5 ± 2.6 g) were housed under the bioterium conditions described above in groups of 6 animals per ventilated cage from postnatal day 30 (P30) until P130. The mice were divided into 4 groups of 12 in each and were treated with intra-gastric gavage daily for 45 days (P30-75) as Group I, 5xFAD mice treated with 1 ml saline (0.9%)/day, a vehicle control group; Group II, 5xFAD mice treated with 200 mg/kg/day W. somnifera, a low dose treatment group; Group III, 5xFAD mice treated with 400 mg/kg/day W. somnifera, a high dose treatment group; and Group IV, 5xFAD mice treated with 200 mg/kg/day Resveratrol (Res, ab120726, Abcam), a positive control group. Following cognitional studies, the brains were dissected out at the mice P130 as previously described 32 . And the brain regions www.nature.com/scientificreports/ associated with AD-related pathology and cognitive decline, namely the frontal cortex, entorhinal cortex, and hippocampus 40,41 , were isolated and used for further studies.
Cognitional studies. To assess the potential in vivo efficacy of the W. somnifera root methanolic extract on the spatial learning and memory of 5xFAD mice, Barnes circular maze and Y-maze spontaneous alternation tasks were used for these procedures require a relatively shorter learning time and involve minimal stress on the mice than the other related paradigms.
Barnes circular maze task. The maze acrylic circular platform (92 cm in diameter with 3 mm thickness) was 91 cm elevated above the floor, consisting of 18 circular holes (9.5 cm in diameter) evenly spaced around the periphery. A detachable acrylic black escape box (12 cm × 23 cm × 12 cm) was attached just under one of the hole's entrance, while the other holes were left open so that the mice cannot enter them. Released from a dark start chamber in the middle of the circular maze after a delay of 10 s each mouse was allowed to explore the maze until it enters the escape box or 5 min elapses for habituation trial, after which the acquisition training phase started in the next 24 h. Following habituation, the mice were trained in spatial learning to escape from the aversive lights by entering the escape box or 3 min elapses. If a mouse fails to enter the escape box within 3 min period, it was gently guided into the escape box and allowed to stay there for 1 min. Training of the mice was carried out in two trials on each day for five consecutive days with an inter-trial interval of approximately 30 min. For each trial, the numbers of error head poke into non-escape holes and the latency time to enter the escape box were recorded to assess performance. Three days after the final session of acquisition training, mice were subjected to a 90 s probe trial to test their spatial learning and memory based on the mice's time-spent in the virtual target-hole zone, the zone which previously contained an escape box. The mice's longer time-spent in the target zone was considered to collectively provide a better indicator of spatial learning and memory.
Y-maze spontaneous alternation task. The maze consisted of three grey non-reflective plastic arms (5 cm × 30 cm × 12 cm) that were all-placed at 120° from each other with partitions. Following the handling habituation session, each mouse was gently placed into the maze center and allowed to explore the arms for a period of 10 min. The mouse number of arm entries and the subsequent number of triads (entries into three consecutive separate arms) were recorded to determine the percentage of spontaneous alternation. An arm entry was defined as the mouse all-four appendages with its snout oriented towards the end of the arm crosses the threshold of the maze central-zone. The spontaneous alternation behavior was determined as the percentage of the number of triads divided by the number of arm entries minus 2, indicated below in Eq. (1). The mice's greater percentage of spontaneous alternation was considered to collectively provide a better indicator of spatial learning and working memory.
Neurochemical studies. Enzyme-linked immunosorbent assay (ELISA). The Aβ 1-42 level was selectively detected using the Aβ 1-42 sandwich ELISA kit. To isolate Aβ 1-42 from the tissue, each mice cerebral cortex and hippocampus were homogenized in ice-cold PBS (0.01 M, pH = 7.4) at 1:9 (tissue weight (g): PBS volume (ml)) and centrifuged at 14,000×g for 30 min at 4 °C. The resulting supernatants were used for the assay. The concentration of Aβ 1-42 in the samples supernatants was determined using the Bradford method 42 , and 100 µl containing 300 µg of proteins from the supernatants fraction were incubated in rabbit anti-mouse Aβ , boiled for 10 min, sonicated, and then cleared by centrifugation at 12,000×g for 10 min. The same amount of lysates was separated by 8% SDS-PAGE gel and electroblotted onto nitrocellulose paper (LC2006, ThermoFisher Scientific). Blots were blocked with 5% nonfat dried milk in 0.1% TBS-T (137 mM NaCl, 0.2% Tween 20, and 20 mM Tris/HCl (pH 7.6)) for 1 h. Immunoblots were incubated overnight at 4 °C with a rabbit anti-NCX3 antibody (1:3000) (ab84708, Abcam) diluted in 1% milk in TBS-T and then incubated with a sheep horseradish peroxidase (HRP)-conjugated anti-rabbit (1:10,000) (ab6795, Abcam) in TBS-T for 1 h. Immunoreactive bands were visualized using the enhanced chemiluminescence system (ECL, ThermoFisher). The optical band intensities were determined by densitometric analysis using Image Lab software, and values were normalized to β-actin (loading control).
Immunohistochemistry. Mouse brain was cut down into transverse coronal sections at 30 µm using a freezing microtome (SLEE, Mainz, Germany). The sections were permeabilized with 0.5% Triton X-100 in PBS for 30 min and then nonspecific protein binding was blocked with 3% bovine serum albumin (BSA) in PBS for 1 h. Then the sections were incubated with the MOAB-2 primary antibody (mouse IgG2 b , 1:1000, ab126649, www.nature.com/scientificreports/ Abcam) overnight at 4 °C. The sections were then washed with PBS (3 × 5 min) and incubated with secondary biotinylated goat anti-mouse at 1:1000 for 1 h. After washing with PBS, sections were incubated with Hoechst 33,342 (10 min) for cell nuclei visualization. Then sections were washed with PBS 3 times, mounted on charged slides, allowed to dry in the dark, and were then incubated with Thioflavin-S staining solution for 10 min. The sections were then dehydrated in a series of alcohols, cleared in xylene, and cover-slipped with 50% glycerin in PBS. Images were acquired using a laser scanning confocal microscope (Zeiss, Tokyo, Japan).
Antioxidant activity assay. Each group representative brain extracts were separately homogenized in PBS (50 mM, pH 7.4) at 1:9 (tissue weight (g): PBS volume (ml)) and centrifuged at 7000×g for 10 min at 4 °C. Then, the supernatant was used as an enzyme source to measure the superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels. Additionally, to measure the glutathione (GSH) level, the group's representative brains were homogenized in glutathione reaction buffer and centrifuged at 10,000×g for 10 min at 4 °C. The supernatants from the centrifuge were collected and immediately treated with sulfosalicylic acid to precipitate the extra proteins and used as enzyme sources to determine GSH level. The amount of protein was quantified by using bicinchoninic acid (BCA) protein assay kit, and the antioxidant activities were assessed using SOD, MDA, and GSH assay kits. The absorbances were measured shortly after the completion of the reaction using a spectrophotometer at 517 nm.
Data analysis. Statistical analysis was performed by using Prism Software (GraphPad 8.0 Software, San Diego, CA). All experiments were performed and analyzed in a blinded manner. The data were evaluated as means ± SEM. Statistically significant differences among means were determined by appropriate statistical tests, including repeated-measures, one-way or two-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. Statistical significance was accepted at the 95% confidence level (p value < 0.05).

Results
The current study W. somnifera roots satisfied the standards of pharmacognostic quality. Physicochemical composition analysis of the W. somnifera roots taken for the current study (loss on drying (2.06%), total ash (4.31%), water soluble ash (1.83%), acid insoluble ash (0.63%), water soluble extractive value (66.62%), and alcohol soluble extractive value (17.34%)), expressed as % of air-dried weight, indicated that the roots satisfy the Ayurvedic Pharmacopoeia of India quality. According to the pharmacognostic standards, the expected values of loss on drying, total ash, water soluble ash, acid insoluble ash, water soluble extractive value, and alcohol soluble extractive value for W. somnifera roots are ≤ 8%, ≤ 7%, ≤ 7%, ≤ 1%, ≥ 15%, and ≥ 15%, respectively 34 . In addition, the analytes on the total sugar, total protein, and total lipid of the roots constitution were determined as 7.15%, 4.24%, and 1.41%, respectively.  (Fig. 1B, C, respectively). Furthermore, analysis of the probe trial data, three days after the final session of acquisition training, provided additional insight on the significant difference between the treatment and vehicle groups in their time spent in the maze virtual target-hole zone, the zone which previously contained an escape box. The result indicated that the treatment groups exhibited a significantly longer time in the target zone compared to the vehicle-treated group (39.5 ± 3.7, 63.6 ± 5.6, and 59.4 ± 6.1 s for the 200 mg/kg/day W. somnifera, 400 mg/kg/day W. somnifera, and Res treatment groups respectively compared to 21.0 ± 3.4 s for the vehicle group), indicative of spatial memory recovery (Fig. 1D). Of note, there were no statistically significant escape behavior performance differences between the www.nature.com/scientificreports/ W. somnifera higher dose (400 mg/kg/day) and Res treated mice during both the acquisition training and probe trial phases of the current study. Moreover, the same murine data obtained from the Y-maze spontaneous alternation task test (Fig. 2) furtherly substantiated the significant efficacy of the W. somnifera roots methanolic extract on spatial learning and working memory of 5xFAD mice. After 45 consecutive days (P30-75) treatment with W. somnifera root methanolic extract, the 200 mg/kg/day-treated and 400 mg/kg/day-treated mice showed remarkable improvement in alternation behavior between the arms of the Y-maze task compared to the vehicle-treated 5xFAD mice (ANOVA followed by Tukey's multiple comparisons test, n = 12 mice/group). Interestingly, there were no statistically significant spontaneous alternation behavior performance differences between the W. somnifera higher dose (400 mg/kg/ day) and the Res treated 5xFAD mice (average of 79.6 ± 4.2% and 75.4 ± 6.0%, respectively).   (in B, C), two-way ANOVA, one-way ANOVA, Tukey's multiple comparisons tests. Significance is indicated by *p < 0.05; **p < 0.01; ***p < 0.001 versus vehicle; by † p < 0.05, † † p < 0.01 between W. somnifera treated groups; by ‡ p < 0.05, ‡ ‡ p < 0.01 between W. somnifera and Res treated groups; by ^p < 0.05 between days 1 and 5 (in C). Vehicle control group, LDT low dose treatment, HDT high dose treatment, Res Resveratrol. Figure 2. Y-maze spontaneous alternation task for spatial working memory test of 4-month-old 5xFAD mice after 45 consecutive days (P30-75) treatment with W. somnifera. Schematic diagram of Y-maze task (A), and data that compare the percentage alternation behavior (B) and their average number of arm entries (C). The data represent the mean ± SEM. Data were from 12 different mice in each group (n = 12) using one-way ANOVA followed by Tukey's multiple comparisons test. Significance is indicated by *p < 0.05; **p < 0.01 versus vehicle; by † p < 0.05 between W. somnifera treated groups; by ‡ p < 0.05 between W. somnifera and Res treated groups. Vehicle control group, LDT low dose treatment, HDT high dose treatment, Res Resveratrol. www.nature.com/scientificreports/ ates of 4-month-old 5xFAD mice brain, immunoblot analysis of the NCX3 expression using a β-actin loading control from the same blot and a sandwich ELISA of the total Aβ 1-42 were performed. Regarding NCX3 protein expression, significantly decreased band levels on the blot were detected both in the cortex (p < 0.01) and the hippocampus (p < 0.001) of the vehicle group compared to those of W. somnifera higher dose (400 mg/kg/day) and Res treatment groups (Fig. 3B, C). The W. somnifera lower dose treatment (200 mg/kg/day) also significantly increased the NCX3 levels both in the cortex (p < 0.05) and the hippocampus (p < 0.05) compared to that of the vehicle group, as observed in the W. somnifera higher dose and Res treatment groups. Noteworthy, a pronounced formation of 75 kDa sized proteolytic fragments were observed in both the cortex and hippocampus of the vehicle group and a few in the hippocampus of the W. somnifera lower dose treatment group, but not in the W. somnifera higher dose and the Res treatment groups (Fig. 3B). Regarding the level of total Aβ 1-42 (Fig. 3D), the quantitative analysis by sandwich ELISA showed a significantly reduced expression of Aβ 1-42 in the cortex and hippocampus of the W. somnifera higher dose (p < 0.05 and p < 0.001, respectively) and Res (p < 0.05 and p < 0.01, respectively) treatment groups compared to that of the vehicle group. Remarkably, while there is no statistically significant difference in the total Aβ 1-42 level in the cortex between the W. somnifera lower dose treatment and vehicle groups, the overall considerably lower level of total Aβ 1-42 in the hippocampus than in the cortex of the W. somnifera treatment groups is worthy of notice. Furtherly as a point of interest, no significant differences in the cortex and hippocampus levels of NCX3 and Aβ 1-42 were observed between the W. somnifera higher dose and the Res treatment groups. These neurochemical study findings suggest that W. somnifera treatment might prevent Aβ 1-42 deposition and the aggregated Aβ 1-42 -induced NCX3 suppression pathways in the cortex and hippocampus of the 5xFAD mice model.

W. somnifera precludes the Aβ aggregation.
To complement the findings obtained from immunoblotting and sandwich ELISA analysis, the effect of the methanolic solution of W. somnifera roots extracts on Aβ plaque deposition, a well-documented earlier pathological characteristic of the 5xFAD mice model 45 , was further investigated using a confocal microscope in the 4-month-old 5xFAD mice-brain, cortex and hippocampus tissues. The confocal microscope immunoreactive studies indicated reduced average number of Aβ plaque deposition and percentage of the area occupied by the plaques in both the cortex and hippocampus tissues of the W. somnifera and Res treatment groups compared to the vehicle group (Figs. 4, 5, ANOVA followed by  Supplementary  Figures S2 and S3. The data represent the mean ± SEM. Data were from 4 different mice in each group (n = 4 of two independent sessions) using one-way ANOVA followed by Tukey's multiple comparisons test. Significance is indicated by *p < 0.05; **p < 0.01; ***p < 0.001 versus vehicle; by † p < 0.05, † † p < 0.01 between W. somnifera treated groups; by ‡ p < 0.05, ‡ ‡ p < 0.01 between W. somnifera and Res treated groups. NCX3 Na + -Ca 2+ exchanger isoform 3, pNCX3 proteolytic fragment of NCX3, Vehicle control group, LDT low dose treatment, HDT high dose treatment, Res Resveratrol. www.nature.com/scientificreports/ Tukey's multiple comparisons test, mean ± SEM, n = 4 mice/group of two independent sessions). Regarding the cortex (Fig. 4), Thioflavin-S staining for Aβ plaque deposition (Fig. 4A) and quantitative analysis on the average number (Fig. 4B) and percentages of the area occupied by the plaques (Fig. 4C) indicated that the W. somnifera higher dose (400 mg/kg/day) and Res treatment groups exhibit notable declines in the number of Aβ plaque deposition (p < 0.05, an average of 24.3 ± 3.2 and 26.4 ± 2.7 plaques/mm 3 for the 400 mg/kg/day W. somnifera and Res treatment groups, respectively, while accumulating to an average of 38.5 ± 1.7 plaques/mm 3 ) and principally in the distribution of the area occupied by the plaques (p < 0.01, 26.5 ± 3.4% and 28.3 ± 3.6% area occupied by the plaques in the 400 mg/kg/day W. somnifera and Res treatment groups, respectively, while distributing to 47.4 ± 1.5%) compared to the vehicle group. However, no statistically significant difference was observed in both the average number of plaques/mm 3 (Fig. 4B) or the percentage of the plaque occupied cortex area (Fig. 4C) between the W. somnifera higher dose and Res treatment groups, and in the average number of plaques/mm 3 between the W. somnifera lower dose treatment and the vehicle groups. As in the cortex of the 4-month-old 5xFAD mice brain, the same groups of murine hippocampus tissue confocal microscope immunoreactive studies exhibited a significant reduction in the average number and distribution of the Aβ plaques/mm 3 of the 400 mg/kg/day W. somnifera and the Res treatment groups compared to the vehicle group (Fig. 5, p < 0.01, ANOVA followed by Tukey's multiple comparisons test, mean ± SEM, n = 4 mice/group of two independent sessions). The reduced production or increased clearance of the Aβ plaque deposition in the hippocampus (Fig. 5B) was significantly higher compared to that of the cortex (Fig. 4B) in the W. somnifera and the Res treatment groups (summary of Figs. 4, 5 confocal microscope images can be found as Supplementary Figure S4). These data suggest that the free radical scavenging activity of W. somnifera 46,47 might have contributed to the oral administration of the extract effects in providing protections against Aβ 1-42 aggregation in the 5xFAD mice cortex and hippocampus. Remarkably, as also observed in the cortex (Fig. 4B, C), no statistically significant difference was observed in the hippocampus tissues on an average number of plaques/mm 3 (Fig. 5B) and the percentage of the plaque occupied area (Fig. 5C) between the W. somnifera higher dose and Res treatment groups.

W. somnifera normalizes oxidative stress. For a further substantiation of the antioxidant effects of W.
somnifera root-methanolic extract and subsequently to elucidate the extract therapeutic mechanism, oxidative stress markers, including MDA, SOD, and GSH, were measured in the 4-month-old 5xFAD mice brain tissue homogenates (Fig. 6). A one-way ANOVA revealed significant differences in the MDA (p < 0.05), as well as both SOD and GSH levels (p < 0.01) compared to that of the vehicle group. The marked increase in MDA level in the Percentage of the area occupied by the plaques. The data represent the mean ± SEM. Data were from 4 different mice in each group (n = 4 of two independent sessions) using one-way ANOVA followed by Tukey's multiple comparisons test. Significance is indicated by *p < 0.05; **p < 0.01 versus vehicle; by † p < 0.05 between W. somnifera treated groups; by ‡ p < 0.05 between W. somnifera and Res treated groups. Vehicle control group, LDT low dose treatment, HDT high dose treatment, Res Resveratrol.

Discussion
Alzheimer's disease (AD) is the only cause of global death in the top ten 48 that cannot be prevented, cured, or even significantly slowed to date. The extracellular aggregation of Aβ 1-42 protein plaques is regarded as a prominent neuropathological characteristic of AD leading to neurodegeneration and cognitive decline [49][50][51] . This aggregation of Aβ 1-42 plaques in the brain is likely caused by aberrant proteolysis of amyloid precursor protein (APP) 51 , and subsequently, the plaques cue calcium-influx-related neuronal damage, including mitochondrial dysfunction, in AD 6,52,53 . For decades, removing the Aβ 1-42 plaques in the brain has been proposed as the principal determinant to prevent AD 50,51,54 . Indeed, numerous studies using AD transgenic mice with aggregated Aβ  plaque evidenced that remotion of these plaques is associated with behavioral improvements 55,56 . In this view, several clinical trials for AD therapeutics have aimed to remove the Aβ 1-42 plaque from the patient's brain [57][58][59] ; however, most of these trials have failed 54,60 . These apparent discrepancies suggest that a long time before the formations of the plaques, small forms of Aβ 1-42 called oligomers appear to be toxic for neurons in such a way that binding of the oligomers to the neurons plasma membrane proteins, such as NCX3, disrupt intraneuronal Ca 2+ homeostasis in AD. Thus the toxic Aβ 1-42 oligomers and their accumulation, plaques, altogether appear to be responsible for AD symptoms. Previous studies of disrupted calcium-influx in AD suggest that aggregation of Aβ 1-42 plaques in neurons may be responsible for NCX3 downregulation observed in the AD pathological changes 3,6 . However, it is unclear whether the renovation of the aggregated Aβ 1-42 induced NCX3 downregulation prevents or delays the onset of AD and AD-related pathology. The present study, for the first time, assessed the therapeutic significance of W. somnifera roots methanolic extract against AD-related Aβ 1-42 aggregation, NCX3 suppression, oxidative stress, and cognitional deficits using 5xFAD mice. W. somnifera has a long history in the traditional medical system as an ingredient in many formulations prescribed for various aging-related health impairments (e.g., arthritis, stress) to prevent disease and improve overall vitality and longevity 9,61 . However, the therapeutic value of this plant roots extract in neurodegenerative diseases, such as AD, albeit far less well studied. To have a baseline of comparison for the effect of W. somnifera in this study, Resveratrol (Res, > 99% purity), a natural polyphenolic compound derived from peanuts, red grapes, blueberries, and other plants [62][63][64] was used as a positive control. Res has extensive records for its neuroprotective features, including inhibition of Aβ plaque formation [65][66][67][68][69][70] and prevention of cognitive impairment 65,66,[70][71][72][73] in AD rodent models. Moreover, due to its antioxidant features 68,70,72 , oral administration of Res is reported to slow aging and increase life span in mice [74][75][76][77] . However, to date, there is no specific report on the potential value of Res to enhance the expression level of membrane transport proteins that involve in the mechanisms of cellular Ca 2+ homeostasis, such as NCX3.
Additionally, an elevated level of Aβ 1-42 has been attributed to induce oxidative stress that further triggers calcium dyshomeostasis in the AD hippocampus and cortex 78 . Oxidative stress, an increase in production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as superoxide radical (O 2 − ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HO − ), nitric oxide (NO), and peroxynitrite (ONOO − ), causes neuronal dysfunction including cell membrane disintegration and mitochondrial damage leading to AD-pathology 79 . To mitigate oxidative stress in AD, several synthetic antioxidants were formulated for clinical trials over the past decades [80][81][82] ; however, there is no Food and Drug Administration (FDA)-approved antioxidant therapy to date for most of the clinical trials raised significant safety concerns. Consequently, alternative natural antioxidants from plant material extracts, such as Res 72 , have recently received much attention. Of note, using an organic solvent in the extraction medium is documented to maximize yield through optimum solvation of components in the milieu and also to enhance favorable influence in the extract phytochemical constituent's activity 83,84 . On these bases, herein, methanol was used as a medium for extractions of W. somnifera roots constituents. This study methanol extraction yielded a mean of 16.49%, expressed as the mass of extract obtained per 100 g of W. somnifera roots, and a yield of 0.27% alkaloid per dry weight of the plant roots, flavonoids content of 67.57 ± 0.97 μg quercetin equivalents/mg, a phenol of 91.09 ± 0.29 μg gallic acid equivalent/mg, and tannins of 35.27 ± 0.51 μg tannic equivalent/mg of the extract (Table 2) which are all consistent with the findings of previously reported studies [84][85][86] . These phytochemical constituents confirm the antioxidant property 86,87 of the W. somnifera roots methanolic extract as a potent to neutralize free radicals and thereby may represent a preventive role against oxidative stress in AD. Moreover, consistent with the observations of previously reported studies on the acute and sub-acute toxicity effects of standardized extracts of W. somnifera roots in rodents 12,14,15 , the current study findings using the daily recorded body weights of the mice (Supplementary Figure S1) during the periods of 200-400 mg/kg/day gavage treatment (P30-75) further ascertains that the W. somnifera roots methanolic extract has no potential toxicity. A noteworthy fact is that a rather intravenous administration enhances efficient delivery of extracts bioactive constituents from the plasma into the blood-brain barrier, which correspondingly this mode of treatment alongside the application of advanced purification technique during the extraction process can substantially decrease the effective high dose used in this study.
After a daily for 45 days, from postnatal day 30 (P30) to P75, oral administration of either 200 mg/kg/day or 400 mg/kg/day methanolic solution of W. somnifera roots extract as a treatment to rescue cognitive impairment, the 4-month-old 5xFAD mice cognition was assessed using the Barnes circular maze task and Y-maze spontaneous alternation task techniques. Barnes circular maze task was performed to examine whether the W. somnifera treated mice display the escape behavior faster than the control mice. The results showed clearly that W. somnifera treated mice could find and enter the escape box significantly faster than their cohort vehicle mice (Fig. 1). According to the analysis of the trend across the Barnes maze sessions in latency, the W. somnifera treated and the Res treated mice continued learning of escape behavior while comparatively not the vehicle mice. The shorter latencies of escape behavior are presumably a result of improved spatial learning and memory in the treated mice, while the exploring behavior toward the other holes looking for the escape box might have increased www.nature.com/scientificreports/ the latency in the vehicles. Allied with this notion, studies on the potential role of Theracurmin 43 , Tyrosol 44 , and Metformin 88 to mitigate AD-related pathology in model mice have adopted a similar protocol. Reassuringly, consistent with the results obtained from the Barnes circular maze task, the same groups of mice Y-maze spontaneous alternation task data proved the potential in vivo efficacy of the W. somnifera roots methanolic extract on the spatial working memory of 5xFAD mice (Fig. 2). Both groups in the daily for 45 days of 200 mg/kg/day and 400 mg/kg/day-treated mice performed better than their cohort vehicle-treated mice on the alternation behavior test. Noteworthy, the alternation behavior of the vehicle-treated 5xFAD mice was consistent with the literature 32 , and there was no statistical difference between the W. somnifera higher dose and the Res-treated mice, implying both treatments were sufficient for behavioral improvement. The Y-maze task behavioral test exploits the innate explorative tendency of rodents to evaluate the potential effects of drugs against the underlying pathology of neurodegenerative diseases, such as AD, on spatial learning and working memory 89 .
Subsequent to cognitional studies, the mice brain regions associated with AD-related pathology and cognitive decline, namely frontal cortex, entorhinal cortex, and hippocampus 40,41 , were isolated and used for further neurochemical studies to determine the potential effects of the W. somnifera roots methanolic extract in each group. Strikingly, in support of the cognitive assay findings, immunoblotting analysis on the expression level of NCX3 and a sandwich ELISA quantification of total Aβ 1-42 level in the cortex and hippocampus homogenates, as well as confocal microscope immunoreactive studies on the Aβ plaque deposition in cortex and hippocampus tissues of the undertaken 4-month-old 5xFAD mice, revealed the neuroprotective significance of W. somnifera roots methanolic extract to rescue neurodegeneration. Regarding the immunoblotting analysis on the expression level of NCX3 (Fig. 3B, C), the determined results indicate that treatment with W. somnifera significantly increases the expression of NCX3 in the cortex and hippocampus. Dysregulation of NCX3, a plasma membrane protein that prominently contributes to the physiological maintenance of Ca 2+ homeostasis in excitable cells, such as neural, has been reported as an early molecular distortion in AD 90,91 . A recent in vitro documented study 3 , using BHK cells that mimic AD, findings indicate the NCX3 neuroprotective effect as confirmed through decreased cell death in NCX3 stably transfected cells (BHK-NCX3) compared to their wild types (BHK-WT), a further correlation between NCX3 and the neuronal death. The NCX3 molecular pharmacology (Fig. 3A) studies 6,92,93 demonstrate that this membrane transport protein contains nine-transmembrane segments (TMS), which exists grouped into two based on their locations in the cytomembrane as an extracellular amino-terminal (NH 2 ) group, the first five TMS, and the intracellular carboxyl-terminal (COOH) group, the last four TMS. These hydrophobic terminal groups, separated by intracellular hydrophilic loop, named as f-loop, are involved in the transport of Ca 2+ across the plasma membrane. The NCX3 f.-loop, critical in the terminals activity regulation, contains two alternative splicing sites recognized as the α-1 repeat, between TMS 2 and 3, and the α-2 repeat, between TMS 7 and 8. It is peculiar to underline that the α-1 and α-2-regions of the f-loop determine the molecular sensitivity of NCX3 94 , and therefore, these regions require extra attention in the pharmacological efforts to develop a compound that targets NCX3. Ordinarily, NCX3 migrates as a 105 and a 75 kDa band in an immunoblot assay (Fig. 3B), whereby these band sizes represent the native NCX3 protein and its proteolytic fragment, respectively 3,90 . Noteworthy, the immunoblot for the NCX3 evidenced that W. somnifera roots methanolic extract enhances the expression level of NCX3 and remarkably prevents fragmentation of this membrane transport protein (Fig. 3B, C). These results suggest that the W. somnifera significance in ameliorating cognitive function may associate with the increased expression level of neuronal membrane NCX3, which is attributable to preventing neuronal cell damage from Ca 2+ dyshomeostasis. Further relative comparison (Fig. 3C) on the observed robust signal expression (Fig. 3B) of the full-length NCX3 protein levels in the cortex than in the hippocampus of the W. somnifera higher dose and the Res treatment groups contrarily revealed a more differential specificity of the treatments benefit to the hippocampal region, suggesting that the effect of the undertaken therapeutic interventions in improving cognitive performance mainly relies on the improved NCX3 regulation in the hippocampus. Additionally, equivalent to the efficacy of the W. somnifera higher dose treatment, the investigated Res treatment effects also showed significant roles against NCX3 dysregulation, which suggests sharing of common attributes among these natural products treatment.
Additional imperative findings from the immunoreactive studies (Figs. 3D, 4,5) are that the oral administration of the W. somnifera roots methanolic extract prevented the Aβ 1-42 aggregation, a well-documented pathological feature of AD 45 , in both the cortex and the hippocampus of 4-month-old 5xFAD mice brain, presumably, through the free radical scavenging activity of the extract phytochemical constituent's. The phytochemical constituents of W. somnifera roots methanol extract (Table 1) include alkaloids, flavonoids, and phenols, which are factual antioxidants 95 . Although the underlying mechanisms of the potent effect of W. somnifera roots extract in Aβ metabolism remain unclear, findings of a reported study using AD transgenic mice also shed light on the extract's ability to enhance efflux of Aβ from the brain to the plasma and eventually lead to its degradation in the periphery 96 . The observed reduction in the Aβ 1-42 deposition and formation of NCX3 proteolytic fragments in the cortex and hippocampus of the treated 5xFAD mice model compared to their cohort vehicle group suggest that W. somnifera treatment might prevent intraneuronal Aβ 1-42 aggregation and the aggregated Aβ 1-42 -induced NCX3 suppression pathways in the AD pathology.
In line with the extract phytochemical screening findings, as expected, oxidative markers, namely MDA, SOD, and GSH levels, were normalized by W. somnifera treatments as evidenced using antioxidant activity assay (Fig. 6). The assay analysis demonstrates that the increased MDA level and decreased SOD and GSH levels in the vehicle-treated 4-month-old 5xFAD mice were significantly reversed by the daily for 45-days (P30-75) administration of 400 mg/kg/day methanolic solution of W. somnifera roots extract, suggesting the pharmaceutical significance of this extract constituents to protect neuronal cells from oxidative damage. Antioxidants have been proposed to neutralize the elevated level of Aβ 1-42 -induced free radicals that causes oxidative stress and Ca 2+ dyshomeostasis in the AD pathology 97 www.nature.com/scientificreports/ antioxidant feature, the herein observed substantial margin of decreased Aβ protein plaque deposition in the treated mice brain regions associated with AD-related pathology compared to that of vehicles further unveils the pharmaceutical significance of W. somnifera roots against AD neurodegeneration. Prevailing basic science translational researches involving various models 20,99-102 and clinical trials 103,104 , on the W. somnifera extracts pharmacological value in improving memory and cognitive function, enlighten several unmet-gaps that require attention for further rigorous investigations to suitably lead to meaningful clinical outcomes, including objectively characterizing the pharmacologically vital constituents of the extract, distinctively determining the molecular mechanisms underlying contributions in the efficacy of the undertaken extract, and using defined and optimized extract constituents to formulate standardized therapeutics strategy for AD. Studies that evaluated the therapeutic remedial activity of the W. somnifera extract, using in vitro and in vivo neurodegeneration model systems, indicate that the extract mechanisms of effects include attenuation of neuroinflammation and increases in the level of autophagic markers in the brain 20,100 , and augmentation of cortical acetylcholine esterase, synaptic plasticity, and neuronal cell survival 101,102 . Further extended clinical trials also highlight the efficacy of W. somnifera extract in moderating age-related memory, information processing, attention and executive function impairments 103,104 , and other clinical benefits that can profoundly impact mental wellbeing, including reducing mental stress and anxiety 105,106 . In considering its Ayurvedic medicinal value, at the moment there is a noteworthy overwhelming computational 107,108 , basic science 109,110 , and clinical research 111,112 momentum and excitement to evaluate the W. somnifera extract in chemoprophylaxis against COVID-19 disease 113 and the long-term effect of COVID-19, post-COVID condition, collateral damage potential contributions in increasing the COVID-19-patients risk of future neurodegenerative diseases, such as AD, where increasing age is a common risk factor in both of these severe conditions, although the effects are not yet explicit. In all, as the investigations looking for an efficient novel therapeutic agent from the plant standardized extracts or bioactive constituents continue, the available research findings strongly suggest considerations to introduce W. somnifera roots in nutritional supplementation regimen formulations as an adjunctive treatment for patients with Alzheimer's neurodegeneration.
In this study context, further studies using NCX3 knockout models would allow for a better understanding of the therapeutic role of W. somnifera against aberrant Ca 2+ influx-induced neurodegeneration, such as AD, as negative controls to uncover novel potential underlying molecular mechanisms of the extract effects in the NCX3 regulation under physiological conditions and during pathologic brain function. Moreover, the chances of observing enhanced progressive improvement in the study mice's cognitive function for the effects of the W. somnifera therapeutic intervention on NCX3 is limited by both the sensitivity and the range of the behavioral tasks yet available. Overall, for the first time, this study reports that W. somnifera ameliorates AD-related pathology, including downregulated NCX3 and cognitive impairment in the 5xFAD mouse model, establishing W. somnifera as a promising candidate to benefit a model with brain integrity challenged by forced amyloidosis created via expression of a mutant human APP transgene.

Conclusions
In summary, the proposed mechanisms of the W. somnifera roots methanolic extract neuroprotective effects against aggregated Aβ 1-42 -induced NCX3 suppression in the 5xFAD mouse model include enhancement of antioxidative activities, a decrease in the Aβ 1-42 aggregation, rectification of NCX3 expression, and likely thereby protection against Ca 2+ dyshomeostasis induced neuronal cell death. These effects of the extract could attribute to the multiple antioxidant phytochemical constituents, and as such, which are likely to have several mechanisms of action. Thus, the W. somnifera roots methanolic extract, with no indications of toxicity, might be regarded for the development of a potential drug candidate in the therapeutics approach or prevention of AD.

Data availability
The data and materials that support the findings of this study are available on request under-identification policy to the corresponding authors. www.nature.com/scientificreports/